1. Field of the Invention
The present invention relates to a method for the biological synthesis of peptides by the genetic manipulation of peptide synthetases.
In particular, the present invention relates to the construction of engineered microorganisms capable of expressing peptide synthetases with a modified substrate specificity.
The present invention also relates to the use of said microorganisms and peptide synthetases extracted from the above microorganisms in the in vivo or in vitro synthesis of biologically active modified peptides.
The term "modified peptides" refers to analogs of wild type peptides or peptides having novel amino acid sequences.
2. Description of the Background
Peptide synthetases are a group of prokaryotic and eukaryotic enzymes involved the a non-ribosomal synthesis of numerous peptides. Non-ribosomal synthesis is also known as thiotemplate synthesis (Kleinkauf, H. and von Doren, H., (1987), Ann. Rev. Microbiol., 41: 259-289).
These peptides (of which several hundred are known) often have non-linear cyclic structures (cyclosporin, tyrocidin, mycobacillin, surfactin and others) or branched cyclic structures (polymyxin, bacitracin and others) and contain amino acids not usually present in proteins as well as D-amino acids.
Peptide synthetases are multienzymatic complexes consisting of one or more enzymatic subunits. The complexes are characterized by the presence of one or more repeated structural units (super-domains) which contain an activation region (DDA) which recognizes a specific amino acid.
Each DDA catalyzes a series of enzymatic reactions, which eventually links each amino acid into a peptidic chain. The enzymatic reactions include:
1-recognition of the amino acids, PA1 2-activation of the amino acids as amino-acyladenylates, PA1 3-binding of the activated amino acid to the enzyme by the thioester bond between the carboxylic group of the amino acid and the SH group of an enzymatic co-factor which is itself bound to the enzyme inside each DDA, and PA1 4-formation of the peptidic bonds among the amino acids.
In addition, the DDA which couple D-amino acids into the peptidic chain also catalyze the racemization of L-amino acids to D-amino acids.
Finally, the multi-enzymatic complex also contains a conserved domain after the last DDA, whose sequence has homologies with various thioesterases, and in particular contains the consensus sequence of their active sites which terminates coupling and releases the peptide product.
In peptide synthetases there are as many DDAs as amino acids which form the peptide. For example the complex of the surfactin synthetase which catalyzes the polymerization of the lipopeptide having the structure: ##STR1## consists of three enzymatic subunits of which the first two include three DDAs each, whereas the third has only one DDA. These domains are responsible for the recognition, activation, binding and polymerization of L-Glu, L-Leu, D-Leu, L-Val, L-Asp, D-Leu and L-Leu.
A similar organization in discrete, repeated units (modules) (see FIG. 1) occurs in the genes encoding the synthetases.
The structure with repeated domains of the peptide synthetases is conserved among the bacterial and fungal species, as shown by the sequence characterization data of the operons srfA (Cosmina et al. (1993) Mol. Microbiol. 8, 821-831), grsA and grsB (Kratzxchmar et al. (1989) J. Bacterial. 171, 5422-5429) tycA and tycB (Weckermann et al. (1988) Nucl. Acid. Res. 16, 11841-11843) and ACV from various fungal species (Smith et al. (1990) EMBO J. 9, 741-747; Smith et al. (1990) EMBO J. 9, 2743-2750; MacCabe et al. (1991) J. Biol. Chem. 266, 12646-12654; Coque et al. (1991) Mol. Microbiol. 5, 1125-1133; Diez et al. (1990) J. Biol. Chem. 265, 16358-16365). Inside the activation domains even of distant species there are sequences with high homology, some of which are conserved and specific for all the peptide synthetases. Other regions, inside the DDA, are more conserved among the DDA which recognize the same amino acid (Cosmina et al. (1992) Mol. Microbiol. 8, 821-831).
The sequence of the peptide produced is determined, at the gene level and therefore at the enzyme level, by the order and sequence characteristics of the single activation domains. This is consequently a basic characteristic of the thiotemplate synthesis system.
It is known that many of the peptides synthesized by the non-ribosomal method have antibiotic, antifungal and immunosuppressive properties and are therefore of considerable commercial importance.
As a result many laboratories are continually looking for molecules with improved properties with respect to the known ones (for example molecules which have immunosuppressive activities similar to those of Cyclosporin A but with less toxicity) or with new activities.
The preparation of new peptide molecules can be carried out using known technology such as by: 1) selecting new host microorganisms, 2) mutagenizing peptide-producing organisms with chemical agents and selecting the mutants which produce a modified peptide, and 3) changing the growth substrates of the peptide-producing organisms to try and favor the incorporation of different amino acids into the peptides.
These methods require a great amount of time, are have a high degree of uncertainty as to the probability of obtaining new molecules and, in addition, offer very few possibilities of directing the design of new peptides.
Another method suggested by existing technology relates to the chemical synthesis of the desired peptides.
The disadvantage of this technology lies in the difficulty of the synthesis, especially for syntheses an a large scale, and above all when the synthesis concerns complex molecules (cyclic, branched structures containing a high number of amino acids or modified amino acids).